Project summary Chronic pain is a debilitating and costly condition from which more than 25 million US citizens suffer on a daily basis. Current available analgesic drugs often do not provide complete pain relief and/or come with serious side effects, and thus, there is a need to develop novel painkillers, that won?t produce restrictive adverse effects. -opioid receptor (OR) agonists are one of the most effective analgesics for moderate to severe acute pain. Yet, the currently available OR agonists come with serious side effects including analgesic tolerance and dependence when used for prolonged periods of time limiting their use primarily to short-term pain treatment or terminal care. This is a major reason why the Center for Disease Prevention and Control created new guidelines recommending against using OR agonists for chronic pain. Targeting ?-opioid receptor (?ORs) instead of ORs has a clear upside as ?OR selective agonists are less addictive, and do not cause problematic constipation and respiratory depression like OR agonists, such as morphine and oxycodone do. Moreover, functional ?OR expression reportedly increases during chronic events, including nerve ligation and persistent inflammation. The OR and ?OR are Gi-coupled receptor that inhibit cAMP production However, these and other G protein-coupled receptors can also signal in a G-protein-independent manner, via ?-arrestin proteins. For opioid receptors recruitment of ?-arrestin is associated with several debilitating adverse effects. Improved understanding of receptor-selective signaling, has made it possible to design pathway-selective agonists, primarily referred to as biased agonists that can avoid or reduce some of these side effects. In the case of ?ORs, strong ?-arrestin recruitment has been associated with seizures, increased alcohol use, hyperlocomotion and analgesic tolerance. Thus, G-protein-biased ?OR agonists hold great potential as novel pain treatment. In this proposal we will combine computational modelling, medicinal chemistry and cellular pharmacology to develop a novel, potent, selective, G-protein-biased ?OR agonist. This proposal is divided in two specific aims: In specific aim 1, we will run molecular dynamics studies on a homology model of an agonist-bound ?OR structure in order to predict binding of G-protein and ?-arrestin biased ?OR agonists. We will use a structure-based approach to rationally design and synthesize novel G-protein-biased ?OR agonists. To speed up the development of the compounds we will use a chemical scaffold that is based on the potent OR agonist drug fentanyl, but importantly can be modified to create ?OR agonist selective agonists. In specific aim 2, we will characterize the synthesized drugs for receptor selectivity, drug potency and signaling bias using specific cellular assays. Analgesic effects of the drugs will be measured in a model of acute inflammatory pain. The obtained information will feed-back to aim 1, to further drive and improve the modelling and synthesis processes. This proposal fits the chemistry science track award for rapid transition R03 mechanism from the National Institute on Drug Abuse, to discover new drugs with low abuse potential.